1. Volume 8, Issue 3, Pages 491-499 (March 2017)
Efficient Precision Genome Editing in iPSCs via Genetic Co-targeting with Selection 
Katie A. Mitzelfelt, Chris McDermott-Roe, Michael N. Grzybowski, Maribel Marquez, Chieh-Ti Kuo, Michael Riedel, Shuping Lai, Melinda J. Choi, Kurt D. Kolander, Daniel Helbling, David P. Dimmock, Michele A. Battle, Chuanchau J. Jou, Martin Tristani-Firouzi, James W. Verbsky, Ivor J. Benjamin, Aron M. Geurts 
Stem Cell Reports 
Volume 8, Issue 3, Pages (March 2017)
DOI: /j.stemcr
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1 Rationale for CTS and Proof of Feasibility
(A) Cells are edited simultaneously with gene-specific CRISPR/Cas9 and ssODN containing a variant of interest (red asterisk) as well as plasmids expressing AAVS1-specific TALENs and a puromycin resistant (PuroR) donor cassette.
(B) Components from (A) are transfected into iPSCs where HDR-receptive cells (green) are more likely to incorporate donor DNA than HDR-refractory cells (red).
(C) Precision-edited versus indel-containing alleles, detected via direct Sanger sequencing of relevant PCR products in hB53 hiPS6 iPSCs following CTS (n = 39) compared with no selection (n = 46) or transient exposure to puromycin (n = 48) (left panel), where n is the number of individual iPSC clones analyzed. Percent of clones bearing the CRYAB:c.325G>C variant (middle panel) and the number of heterozygous/homozygous clones (right panel).
(D) Representative chromatograms showing local CRYAB sequence of a wild-type (WT) clone (top) and that of a clone bearing a CRYAB:c.325G>C (homozygous) knockin allele (black arrow, c.325G>C variant; gray arrow, Cas9-blocking silent variant). See also Figures S1, S2, S4 and Table S1.
Stem Cell Reports 2017 8, DOI: ( /j.stemcr )
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3. Figure 2
Quantitative Analysis of CTS-Enabled Precision Editing Across Multiple Genes
(A and B) Representation of precision (HDR only) editing events, based on read sequence and normalized to total read count, with (+) and without (−) CTS at multiple loci in hB53 hiPS6 (A) and hB119 hiPS9 (B) iPSC lines (−) CTS indicates cells handled in the same way as (+) CTS except for the addition of puromycin.
(C) Average fold change in HDR-mediated knockin with CTS relative to that without CTS, considering all loci in hB53 hiPS6 and hB119 hiPS9 iPSC lines.
(D and E) Relative proportions of reads bearing NHEJ- and HDR-based edits with (+) and without (−) CTS at multiple loci in hB53 hiPS6 (D) and hB119 hiPS9 (E) iPSC lines.
(F) Average increase in the HDR/NHEJ ratio in hB53 hiPS6 and hB119 hiPS9 iPSC lines with CTS relative to without CTS. See also Figure S3 and Table S2.
Stem Cell Reports 2017 8, DOI: ( /j.stemcr )
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4. Figure 3 Validation of Knockin Cell Lines Generated with CTS
Knockin clones were generated for each disease-associated variant of interest shown in Table S3, with representative clones harboring variants in each gene shown here. Chromatograms (chromats) showing Sanger sequencing results of original cell line (WT) and knockin line (KI) with variants indicated by black arrows. Immunocytochemistry showing pluripotency markers Nanog and SSEA-4 for each knockin cell line harboring the respective variant of interest. Images were merged and counterstained with DAPI. Scale bar, 100 μm. Representative karyotypes for each cell line. Agarose gel showing AAVS1 inside-out PCR for both the 5′ (middle lane) and 3′ (right lane) integration sites (Experimental Procedures), which demonstrates site-specific integration of the selection construct via HDR. See also Figure S3 and Table S3.
Stem Cell Reports 2017 8, DOI: ( /j.stemcr )
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5. Figure 4 Simultaneous Dual Modification using CTS
(A) Dual modification was attempted on hB53 hiPS6 using components for targeting MTERF4:c.693delATA and CRYAB:c.358A>G. Following CTS, 50 clones were sequenced and, of those, 14 had knockin at CRYAB (depicted in the red circle) and 6 had knockin at MTERF4 (depicted in the blue circle). All 6 of the cells with MTERF4 knockin also had knockin at CRYAB. Given individual editing rates, the likelihood of co-occurrence assuming random distribution of events is 5%. We observed a disproportionate co-occurrence of dual modification with a FET <
(B) Representative chromatograms of dual-targeted clones at each loci and WT sequence. Black arrows indicate variant of interest position. Silent, engineered blocking mutations that prevent re-targeting by Cas9 are indicated by gray arrows.
(C) Table including genotypes of individual clones at both (MTERF4 and CRYAB) loci. WT, unmodified; KI, knockin; Indel, insertion or deletion. See also Table S3.
Stem Cell Reports 2017 8, DOI: ( /j.stemcr )
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6. Stem Cell Reports 2017 8, 491-499DOI: (10.1016/j.stemcr.2017.01.021)
Copyright © 2017 The Authors Terms and Conditions